DNA Cleavage 41 Active Sites

Homing endonucleases cleave their double-stranded substrate DNAs at positions +2/+3 and -2/-3 in the presence of Mg2+ and other divalent ions to generate cohesive four-base extension overhangs. DNA cleavage activity has not been demonstrated for all isolated inteins that harbor associated endonuclease domains. Some intein-endonucleases fail to cleave DNA due to accumulated mutations that affect binding or catalysis. The catalytic center of homing endonucleases consists of two overlapping active sites, each defined by a conserved aspartate, that are located at the end of the LAGLIDADG helices (Christ et al. 1999). The aspartates coordinate the divalent metals that act as cofactors in DNA cleavage (Schottler et al. 2000). Other divalent metals can substitute for Mg2+ but not Ca2+ which acts as competitive inhibitor of the hydrolytic reaction. Mn2+ relaxes the specificity of some inteins, such as Pl-Scel (Gimble and Thorner 1992), and rescues the activity of mutants with reduced activities.

When superimposed, the active sites of homing endonucleases show no homology at the primary level except for the catalytic aspartates which bind the essential Mg2+ ions and one or two lysines which are also related by a symmetric or pseudo-symmetric two-fold axis (Fig. 3). The lysines have been proposed to act as general bases in the catalytic mechanism. In fact, in I-Crel, Lys 98 and Lys 98' have been found to be critical for catalysis (Seligman et al. 1997), whereas in Pl-Scel only one of them, Lys 301, seems to be essential for cleavage activity (Gimble et al. 1998). Mutations of nearby residues such as Asp 229 and its pseudo-symmetry related His 343 in Pl-Scel (Gimble and Wang 1996) and Gin 47 and Gin 47' in I-Crel (Seligman et al. 1997) also decrease cleavage activity. These residues are not conserved among the active sites of homing endonucleases and inteins whose structures have been solved (Fig. 3), indicating the high divergence of this family. Their participation in hydrogen-bonding water networks, which are likely to vary depending on the protein, might explain their function in catalysis.

4.2 Divalent Metal Binding at the Active Sites

The DNA-bound structure of the homodimeric I-Crel was the first to reveal the presence of three metals at the active sites that are symmetrically distributed (Fig. 3) and are coordinated to the aspartates and scissile phosphates (Chevalier et al. 2001). One of the metals (number 3) is shared between the two aspartates, suggesting two overlapping active sites with a two-metal mechanism, similar to the two-metal mechanism of some restriction endonucleases (Galburt and Stoddard 2002). A water molecule bound to each of the unshared metals acts as nucleophilic water since it is absent in the structure of I-Crel bound to its product. The identity of the general base that abstracts a proton from the nucleophilic water is not known. Instead, the water network that connects the nucleophilic water to nearby residues which are important for cleavage and to the leaving group was proposed to act as a nucleophile. The monomeric I-Scel (Moure et al. 2003) also has three metal ions at the active sites, two unshared and one shared between the aspartates, but, in contrast to I-Crel, they are arranged in an asymmetric manner (Fig. 3). The asymmetric arrangement of the metal ions is due largely to the asymmetric bending of the top and bottom DNA strands resulting in the bottom strand being closer to the active sites. Consequently, the unshared metal ion that coordinates the scissile phosphate of the top strand is buried deep into the minor groove of the DNA and is not coordinated to the either of the aspartates. The PI-SceI/DNA/Ca2+ ternary structure was obtained at 3.5 A resolution (Moure et al. 2002). Despite the low-resolution structure, difference electron density maps clearly showed the distinctive presence of two peaks in the active sites that correspond to two divalent metals that are 4.2 A apart. The structure of the homing endonuclease and maturase I-Anil also shows two metals at the active sites (Bolduc et al. 2003).

The differences in the number of metal ions and their locations as well as the presence of dissimilar DNA conformations (Fig. 3) and water networks at the active sites suggest divergence in DNA-cleavage mechanisms. For instance, I-Crel, which cannot discriminate between the top and bottom strands of its DNA substrate, cleaves both strands at the same rate which is reflected in the symmetry of the active sites. In contrast, I-Scel, which preferentially cleaves the bottom strand, shows an asymmetric arrangement of metals ions in which the bottom strand cleavage site seems ready to be cleaved first (Moure et al. 2003). The metal bound to the top strand cleavage site is coordinated to DNA bases via water molecule interactions, indicating the possibility of substrate-assisted catalysis. For Pl-Scel, the two metals found at the active sites are colinear with the scissile phosphate of the top strand, suggesting that the top strand might be cut first. Although no nicking activity has been detected for Pl-Scel, mutational and metal mapping studies (Christ et al. 1999) are consistent with a preference for top strand cleavage. Biochemical experiments show that some inteins (e.g. Pl-Pabll) cut the DNA in a concerted manner (Saves et al. 2002), while others (e.g. Pl-Pful, PI-MtuI, Pl-PabI, and PI-Tful) cut the DNA in a sequential mode (Ichiyanagi et al. 2000; Guhan and Muniyappa 2002; Saves et al. 2002; Thion et al. 2002). In the apo structure of Pl-Pful, the absence of the residue equivalent to Pl-Scel's Lys 301 results in two non-equivalent catalytic sites, which the authors suggest as the source of different rates of cleavage for the top and bottoms strands (Ichiyanagi et al. 2000). It is also possible that all homing endonucleases use a similar mechanism in which both strands are cut simultaneously but with different efficiencies by the two active sites.

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